目的 探讨泛素连接酶肌肉环指状蛋白2(muscle ring finger protein 2,MuRF2)对缺氧心肌细胞活力和自噬的调控作用.方法 构建大鼠源MuRF2基因过表达和敲减慢病毒载体,分别感染大鼠心肌细胞H9C2.将H9C2细胞分为阴性对照组(NC)、阳性对照组[MuRF2过表达空载体组(LV-Vector)、MuRF2敲减空载体组(LV-RNAi-Vector)]、MuRF2 过表达组(LV-MuRF2)和 MuRF2 敲减组(LV-RNAi-MuRF2),缺氧培养(5％CO2、1％02、94％N2)后,采用CCK-8法和ELISA法检测心肌细胞损伤水平,利用透射电子显微镜检测心肌细胞超微结构和自噬水平变化,采用Western blot测定自噬相关蛋白LC3-Ⅱ和P62的表达水平.结果 与对照组相比,缺氧组心肌细胞间隙增大、细胞皱缩、多见漂浮的死亡细胞和细胞碎片,心肌细胞活力下降、肌钙蛋白Ⅰ(cTn Ⅰ)含量增加(P均＜0.05);电子显微镜下可观察到部分线粒体发生肿胀,并存在嵴断裂、嵴溶解现象,自噬小体散在胞质内;缺氧组LC3-Ⅱ蛋白表达水平升高,P62蛋白表达水平降低(P均＜0.05).缺氧培养后,与NC组和LV-Vector组相比,LV-MuRF2组细胞活力增加,偶见自噬小体,LC3-Ⅱ蛋白表达水平降低、P62蛋白表达水平增高(P均＜0.05);与NC组和LV-RNAi-Vector组相比,LV-RNAi-MuRF2组细胞活力降低,可见大量自噬小体,LC3-Ⅱ蛋白表达水平升高、P62蛋白表达水平降低(P均＜0.05).结论 泛素连接酶MuRF2可减轻缺氧所致的心肌细胞损伤,其机制可能与抑制心肌细胞线粒体自噬有关.
Abstract
Objective To define the regulatory effects of ubiquitin ligase muscle ring finger protein 2(MuRF2),a ubiquitin ligase,on the viability and autophagy of hypoxic cardiomyocytes.Methods Rat-original lentivirus vectors of MuRF2 overexpression and knockdown were constructed and infected rat cardiomyocytes H9C2 respectively.The H9C2 cells were divided into negative control(NC)group,positive control(LV-Vector、LV-RNAi-Vector)group,MuRF2 overexpression group(LV-MuRF2),and MuRF2 knockdown group(LV-RNAi-MuRF2).After hypoxic(5％CO2,1％O2,94％N2)culture,cardiomyocyte damages were assessed using the CCK-8 and ELISA assays.Changes in the ultrastructure and autophagy of cardiomyocytes were examined using transmission electron microscopy.The expression levels of autophagy-related proteins LC3-Ⅱ and P62 were de-termined by Western blot.Results Compared with the control group,the hypoxia group exhibited increased in-tercellular gap,cellular shrinkage,and a higher presence of floating dead cells and cell fragments;decreased my-ocardial cell viability,elevated levels of cardiac troponin Ⅰ(cTn Ⅰ)(P all＜0.05).Electron microscopy revealed swollen mitochondria with phenomena such as cristae disruption and dissolution,along with scattered autophagic vacuoles in the cytoplasm.The hypoxia group showed increased expression of LC3-Ⅱ protein and decreased ex-pression of P62 protein(P all＜0.05).After hypoxic culture,compared with the NC group and LV-Vector,cells overexpressing MuRF2 showed increased viability,occasional autophagosomes,de-creased expression levels of LC3-Ⅱ protein,and increased expression levels of P62 protein(P all＜0.05);compared with the NC group and LV-RNAi-MuRF2 group,cells with MuRF2 knockdown exhibited decreased viability,numerous autophagosomes,increased expression levels of LC3-Ⅱ protein,and decreased expression levels of P62 protein(P all＜0.05).Conclusion Ubiquitin ligase MuRF2 alleviates hypoxia-induced cardiomy-ocyte damage,and its role in mitophagy inhibition may be one of the underlying mechanisms.
关键词
泛素连接酶/肌肉环指状蛋白2/心肌细胞/缺氧/线粒体自噬
Key words
ubiquitin ligase/muscle ring finger protein 2/cardiomyocytes/hypoxia/mitophagy
维普
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